Functionalization of lactose as a biological carrier for bovine serum albumin by electrospraying
Graphical abstract
Introduction
Particle technology (the science and technology related to the handling and processing of particles and powders; Rhodes, 2008) plays a key role in the development of new formulations for drug delivery systems (DDS). The small quantities of the active principle needed for each dosage are very difficult to evenly distribute in the solid state (Islam and Gladki, 2008). In the case of pulmonary delivery, an additional problem is that only a small fraction of the drug will reach the lungs and most of it will remain in the throat of the patient. This is particularly important when dealing with expensive molecules like proteins and peptides (Service, 1997, Wurm, 2004, Demain and Vaishnav, 2009). The most common drug delivery route for proteins is direct parenteral (intravenous, intramuscular, subcutaneous) which is inconvenient, painful and unsafe. Ideally, the use of non-invasive delivery method such as oral administration is suitable, however, proteins show a poor adsorption due to their large size and hydrophilicity and the gastro-intestinal tract environment promotes their extensive degradation. Thus, the development of new formulations based on particle technology, aims new DDS able to cross particular physical barriers, in order to better target the drug and improve its effectiveness, or on finding alternative and acceptable routes for the delivery of proteins drug (Rao et al., 2008). For these molecules and despite the restrictions associated with the use of pressurized metered dose inhalers (pMDIs) (Newman and Busse, 2002), the preferred option is still the use of dispersed aqueous solutions to deliver the proteins into the lungs (Service, 1997). The reasons for this are the difficulties associated with the use of the conventional techniques for particle size reduction like milling, recrystallization from liquid antisolvents, freeze-drying or spray drying (Subramaniam et al., 1997). Although these micronization techniques are very efficient in the processing of small molecules with highly crystalline morphologies, the disruptive nature of these processes make them inadequate to work with labile biological molecules, with complex structures, such as porous/hollow particles, non-spherical particles, composites, nano-aggregates and surface-modified materials (coated or encapsulated) (Chow et al., 2007). A good alternative for the production of nano- and micron-sized particles of biological active molecules is electrohydrodynamic atomization (EHDA), a process in which a liquid jet breaks up into droplets under the influence of electrical forces (Ciach, 2006, Grace and Marijnissen, 1994, Cloupeau and Prunet-Foch, 1994). A liquid is pumped through a nozzle at a low flow rate (10−6 L/h–10−3 L/h). An electric field is applied over the liquid by applying a potential difference between the nozzle and a counter electrode. When the electric stresses overcome the surface tension stresses, the emerging liquid meniscus from the tip of the nozzle is transformed into a conical shape. From the cone apex a jet emerges which breaks up into quasi-monodisperse droplets. Unipolarity of the droplets prevents their coagulation and dispersion is enhanced (Jayasinghe et al., 2002). Droplets of a few micrometers, or even smaller, can be obtained, which results in particles with a very narrow particle size distribution in the desirable range (Jayasinghe and Edirisinghe, 2002, Gomez et al., 1998, Yurteri et al., 2010). This process was already successfully applied to some proteins like insulin (Pareta et al., 2005) and bovine serum albumin (BSA), both pure (Xu and Hanna, 2007), encapsulated with chitosan (Elversson et al., 2002) or just in the production of chitosan solid micro and nanoparticles (Zhang and Kawakami, 2010). These last examples show how the versatility of this technique allows developing more complex formulations for biological active compounds. On the other hand, the properties of an excipient can improve the formulation by reducing the risk of local overdose, by allowing for more accurate dosage and by giving an additional control over the pharmacokinetics. However, simply mixing active ingredient with the excipient may lead to mixing and sub-optimal dosing. If we use instead a composite where the active molecule is physically attached to the excipient, it presents mainly the physical properties of the excipient. Only when the excipient is dissolved in the mucus, the protein is released and can act. This simple concept opens the door to several applications of these composites. In this work, a new composite was developed where the lactose, a commonly used excipient for pulmonary delivery (Zijlstra et al., 2004, Sambrook, 2001) was coated with BSA by electrohydrodynamic atomization (van Ommen et al., 2010). The obtained powder was analysed for concentration, morphology, particle size and biological activity to access the possibility of using this new formulation for pharmaceutical and biological applications.
Section snippets
Materials
The lactose used in this work consisted of the commercial formulation LactoHall Crystals from DOMO, Friesland Foods (The Netherlands) (d(10) = ±77 × 10−6 m; d(50) = ±123 × 10−6 m and d(90) = ±175 × 10−6 m). The BSA (≥96%) and the acetic acid (>98%) were purchased from Sigma–Aldrich (The Netherlands). The dimethyl sulfoxide, DMSO, (≥99.9%) was supplied by Merck (Germany) and the ethanol (≥96%) was obtained from Brentag Nederland BV (The Netherlands).
Electrohydrodynamic atomization (EHDA)
The EHDA experiments were carried out in a setup
Influence of the solvent in EHDA
The first choice regarding solvents for this type of molecules should always be water, since these are biological systems and if one thinks about industrial applications, water is always easier and less expensive to deal with. Although water was already used as solvent for the electrohydrodynamic atomization (EHDA) of BSA (Pareta et al., 2005), its use in this case is not suitable since the goal of this work was the application of the technique coupled with BSA deposition on the surface of
Conclusion
A new formulation consisting on a composite of lactose and BSA was successfully produced by electrohydrodynamic atomization (EHDA), where BSA particles with mean diameters around 700 × 10−9 m were adsorbed at the surface of lactose crystals by means of electrostatic forces. Biological tests have shown that the protein does not suffer degradation or significant conformational changes with the process. This new formulation can be tested for pulmonary delivery of proteins, giving an additional
Acknowledgements
The authors would like to thank DOMO – Friesland Foods (The Netherlands) for gently supplying the lactose used in this work. D. Harkema is also acknowledged for her collaboration in the experimental work. P.A.M.H. Soares thanks FCT (Portugal) for the research grant ref. SFRH/BD/47740/2008.
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